Regulation of normal and Leber congenital amaurosis-associated RPE65s

正常和 Leber 先天性黑蒙相关 RPE65 的调节

• 批准号: 8184667
• 负责人: Minghao Jin
• 金额: $ 35.5万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8184667
• 关键词: 11 cis Retinal; 11-cis-Retinol; 26S proteasome; Acyl Coenzyme A; Binding; Biochemical; Biochemical Reaction; Blindness; Cattle; Cells; Childhood; Coenzyme A Ligases; Cyclic GMP; Degenerative Disorder; Development; Enzymes; Esters; Exhibits; Expression Library; Gated Ion Channel; Genes; Goals; Human; Hyperactive behavior; In Vitro; Isomerase; Isomerism; Knock-in Mouse; Knock-out; Knowledge; Lead; Leber' s amaurosis; Light; Link; Membrane; Membrane Proteins; Missense Mutation; Molecular; Mus; Mutant Strains Mice; Mutation; Natural regeneration; Opsin; Pathogenicity; Pathway interactions; Patients; Phenotype; Photoreceptors; Phototransduction; Pigments; Proteins; RPE65 protein; Regulation; Research; Research Project Grants; Retinal Degeneration; Retinal Pigments; Retinoids; Rhodopsin; Role; Screening procedure; Series; Signal Transduction; Site; Structure of retinal pigment epithelium; Testing; Therapeutic Intervention; Ubiquitin; Very Long Chain Fatty Acid; Vision; base; cold temperature; early onset; expression cloning; fatty acid-transport protein; in vivo; inhibitor/antagonist; innovation; loss of function; multicatalytic endopeptidase complex; mutant; novel; photoreceptor degeneration; prevent; response; retinol isomerase; retinyl palmitate; visual cycle

DESCRIPTION (provided by applicant): The visual cycle is a series of enzymatic reactions essential for regenerating 11-cis retinal (11cRAL), which functions as a molecular switch for activating opsin in response to light stimulation. 11cRAL keeps opsin inactive. When light hits the visual pigments, its energy converts the pigments' 11cRAL into all-trans isomer, thus activating opsin. The activated opsin then triggers phototransduction, which converts the light-activated biochemical signal to an electrical energy by closing the cGMP-gated ion channels in the photoreceptors. Since the opsin that lost 11cRAL is no longer responsive to light, 11cRAL must be regenerated via the visual cycle to re-form the light-sensitive visual pigments. The key enzymatic reaction in the visual cycle is the isomerization of the all-trans retinoid to an 11-cis isomer. We have identified RPE65, a retinal pigment epithelium (RPE) specific membrane-associated protein, as the retinoid isomerase. Mutations in the human RPE65 gene have been associated with an early-onset retinal degenerative disease known as Leber congenital amaurosis (LCA). Despite the importance of this protein, the molecular mechanisms that regulate the function of RPE65 are largely unknown. The long-term goals of our research are (1) to define the mechanisms that regulate the enzymatic activity of normal and LCA-associated (LCAA) RPE65s and (2) to develop a novel and effective strategy for rescuing LCAA RPE65s. Identification and characterization of proteins that regulates RPE65 function is the key to defining the mechanisms. By screening of the bovine RPE expression library, we have identified three negative regulators of RPE65. The goal of Specific Aim 1 of this proposal is to elucidate the action mechanisms of the negative regulators in vitro and determine their functional role in the visual cycle in vivo. As a step toward defining the inhibitory mechanisms of RPE65 by the regulators, we will first test if the new regulators bind with RPE65. Next we will test if the new regulator can compete with RPE65 for binding to the substrate of RPE65. We will then determine the role of the new regulator in vision by analyzing the visual cycle-related phenotypes of the mutant mice that lack the new regulator. The goal of Specific Aim 2 of this project is to determine whether the new regulators are involved in the pathological mechanism of the LCAA RPE65 mutations and to define the molecular basis for the pathogenicity of LCAA RPE65s with non-active site missense mutations. The proposed research is innovative because it can establish a new research direction in regulation of isomerase activities of normal and LCAA RPE65s and can lead to the development of a novel rescue strategy for delaying or preventing vision loss and photoreceptor degeneration in patients with LCA that is caused by mutations in the RPE65 gene. PUBLIC HEALTH RELEVANCE: Relevance: Mutations in the human RPE65 gene cause early onset childhood blindness known as Leber's congenital amaurosis (LCA). The proposed research project will increase our understanding of how the function of the normal and LCA-associated mutant RPE65s is regulated. The knowledge gained from this research will facilitate the development of a novel and effective therapeutic intervention to delay or prevent vision loss in patients with LCA.

描述（由申请人提供）：视觉循环是再生 11-顺式视网膜 (11cRAL) 所必需的一系列酶促反应，11cRAL 充当响应光刺激而激活视蛋白的分子开关。 11cRAL 使视蛋白保持不活跃。当光线照射到视觉色素时，其能量将色素的 11cRAL 转化为全反式异构体，从而激活视蛋白。然后，激活的视蛋白触发光转导，通过关闭光感受器中的 cGMP 门控离子通道，将光激活的生化信号转换为电能。由于失去 11cRAL 的视蛋白不再对光做出反应，因此 11cRAL 必须通过视觉循环再生，以重新形成光敏视觉色素。视觉循环中的关键酶反应是全反式类维生素A异构化为11-顺式异构体。我们已将 RPE65（一种视网膜色素上皮 (RPE) 特异性膜相关蛋白）鉴定为类视黄醇异构酶。人类 RPE65 基因突变与一种称为莱伯先天性黑蒙 (LCA) 的早发性视网膜退行性疾病有关。尽管这种蛋白质很重要，但调节 RPE65 功能的分子机制在很大程度上尚不清楚。我们研究的长期目标是 (1) 确定调节正常和 LCA 相关 (LCAA) RPE65 酶活性的机制，以及 (2) 开发一种新颖且有效的策略来拯救 LCAA RPE65。调节 RPE65 功能的蛋白质的鉴定和表征是定义机制的关键。通过对牛RPE表达文库的筛选，我们鉴定出了RPE65的三个负调控因子。该提案的具体目标 1 的目标是阐明体外负调节因子的作用机制，并确定它们在体内视觉循环中的功能作用。作为调节剂定义 RPE65 抑制机制的一步，我们将首先测试新的调节剂是否与 RPE65 结合。接下来我们将测试新的调节剂是否可以与 RPE65 竞争与 RPE65 底物的结合。然后，我们将通过分析缺乏新调节器的突变小鼠的视觉周期相关表型来确定新调节器在视觉中的作用。该项目的具体目标2的目标是确定新的调控因子是否参与LCAA RPE65突变的病理机制，并确定具有非活性位点错义突变的LCAA RPE65致病性的分子基础。该研究具有创新性，因为它可以为正常和LCAA RPE65s异构酶活性的调节建立一个新的研究方向，并可以导致开发一种新的救援策略，以延缓或预防由RPE65基因突变引起的LCA患者的视力丧失和光感受器变性。 公共卫生相关性： 相关性：人类 RPE65 基因突变会导致儿童早发性失明，称为莱伯先天性黑蒙 (LCA)。拟议的研究项目将增加我们对正常和 LCA 相关突变 RPE65 的功能如何调节的理解。从这项研究中获得的知识将有助于开发一种新颖有效的治疗干预措施，以延缓或预防 LCA 患者的视力丧失。